Approximately 3% of the world population is infected with the Hepatitis C virus (HCV). As a major cause of serious liver disease, HCV represents an urgent public health problem. Licensed antiviral therapies are non-specific and there is currently no vaccine against HCV. It has been shown that the humoral response predominantly target the E2 envelope glycoproteins. In order to escape from recognition by neutralizing antibodies, HCV has evolved glycosylation sites that obscure functionally critical sites on E2. These glycans also probably attenuate the ability of the immune system to effectively elicit neutralizing antibodies against the critical epitopes that they obscure. We recently showed that removal of certain glycosylation sites in the HCV E2 envelope glycoprotein generates a virus that is hyper-sensitive to neutralization by HCV+ patient sera. Two of the glycans were found to mask the highly conserved CD81 coreceptor binding site. We hypothesize that immunizing mice with these E2 glycosylation mutants, coupled with strategies that non-specifically boost innate immunity, will focus the humoral response on the CD81 binding site thereby eliciting high titers of broadly reactive neutralizing antibodies. Such antibodies are rare when elicited by wild type E2 wherein the CD81 binding site is occluded. In Specific Aim 1 we will immunize mice with glycosylation mutants of the recombinant soluble E2 protein formulated in various adjuvants. The resulting immune sera will be characterized for potency and breadth of neutralization. In Specific Aim 2 neutralizing monoclonal antibodies (MAbs) will be derived from animals with the highest neutralizing serum titers. Combinations of MAbs will be tested for synergy in their ability to inhibit viral entry. We will also determine whether neutralization correlates with MAb inhibition of E2-CD81 binding. In Specific Aim 3 we will determine which neutralizing sera, MAbs or combinations of MAbs can attenuate or prevent outgrowth of viral escape variants. Our work will have broad implications for designing vaccines to specifically block HCV infection. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) is a leading cause of life-threatening liver disease. We seek to design candidate vaccines to specifically block HCV infection.